PROJECT SUMMARY/ABSTRACT Despite the fact that Heart Failure with Preserved Ejection Fraction (HFpEF) accounts for >50% of HF in the U.S., there are no effective therapies for this disease, in contrast to HF with Reduced Ejection Fraction (HFrEF). Similarly, while studies of Ca regulation and excitation-contraction (EC) coupling in HFrEF have led to important therapies to improve contractile function and reduce arrhythmia, the same cannot be said for HFpEF. The lack of fundamental understanding of Ca regulation in HFpEF is a major impediment to the development of rational and effective therapies for this disease in humans. We propose to use the Dahl Salt-sensitive (DS) rat model of hypertension-induced HFpEF, which exhibits the major hemodynamic and clinical features of human HFpEF, to identify and investigate distinctive features of cellular Ca regulation, EC coupling and myofilament Ca response, which influence contractility, diastolic relaxation and rhythm. Results will be compared with a complementary rat model of post-infarction HFrEF. Our goal is to test the overall hypothesis that abnormalities in Ca regulation, not previously demonstrated in a phenotypically-verified HFpEF rat model, are not only distinct from HFrEF but also collectively contribute to the hemodynamic and electrophysiological abnormalities that characterize HFpEF. We have three aims: Aim 1: EC Coupling & Contractility in HFpEF vs HFrEF: In this aim, we will use patch clamp and simultaneous confocal imaging of Ca dynamics to test the hypothesis that Ca-induced Ca release (CICR) is markedly enhanced in HFpEF by changes in the activity of specific Ca-regulating ion channels and transporters residing in the cardiac couplon, including Ca and Na channels, sodium-calcium exchange (NCX) and ryanodine receptors. Aim 2: Diastolic Calcium & Ventricular Relaxation in HFpEF vs HFrEF: We will test the overall hypothesis that the key abnormality of diastolic Ca regulation in HFpEF, i.e. defective SR Ca reuptake and NCX Ca efflux, is only apparent upon ?-adrenergic stimulation, whereas Ca uptake is abnormal at rest in HFrEF. We will use live confocal imaging to dissect the role of Ca uptake mechanisms (e.g. SERCA) and extrusion (e.g. NCX) in the declining phase of the Ca transient, and assess the effects of posttranslational modifications identified by our proteomics screen. Myofilament assays will reveal the extent whereby changes in Ca binding properties and force generation versus passive stiffness contribute to abnormal relaxation. Aim 3: Disturbances of Atrial & Ventricular Rhythm in HFpEF vs HFrEF: Using implantable telemeters, whole heart dual voltage and calcium mapping, and single sinoatrial node (SAN) cell patch clamp, we will test the hypothesis that abnormal Ca regulation plays a critical role in chronotropic incompetence and arrhythmia in HFpEF. We will focus our studies on the role of altered SAN gene expression indicated by next generation RNA sequencing. In conclusion, this project will provide an advanced understanding of the differences between Ca regulation in HFpEF and HFrEF that may generate novel HFpEF therapies based on that entity?s unique cellular physiology.